Systems and methods for the digestion of adipose tissue samples obtained from a client for cryopreservation

ABSTRACT

The present invention is directed to systems and methods of processing aspirated adipose tissue for the isolation of stromal vascular fraction derived stem cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This is continuation application which claims priority to U.S.application Ser. No. 13/646,647 filed Oct. 5, 2012 under 35 USC 120,incorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and systems for digestion of adiposetissue samples for the isolation of the stromal vascular fractioncontaining viable, uncontaminated stem cells, obtained from a client forcryopreservation.

BACKGROUND OF THE INVENTION

Conventional adipose tissue extraction methodologies and devicestypically used within research formats to study, develop, and summarizetheories and or conclusions, are unsuitable for clinical application.Because of the animal derived products and non-cGMP (current goodmanufacturing practices) materials used in laboratory techniques duringthe handling and processing, use of the final products would beimpermissible for human application. Advancements in tissue handling,manipulation, and processing techniques are being developed for theimmediate clinical application of the processed adipose tissuematerials.

In recent years, the discovery of adipose-derived stem cells in thestromal vascular fraction (SVF) of adipose tissue, specificallymesenchymal stem cells, have led to advances in tissue re-growth anddifferentiation. The SVF is that portion of adipose tissue other thanthe mature adipocytes, which can be separated after enzymatic digestionof the tissue to release individual cells from the extracellular matrix,followed by centrifugation. This fraction contains monocytes (whiteblood cells), erythrocytes (red blood cells), mesenchymal stem cells,committed preadipocytes, microvascular (capillary) endothelial cells,and endothelial progenitor cells. A particular future use ofadipose-derived stem cells—the stromal vascular fraction, more specificto mesenchymal stem cells, could be the enhancement of the body'snatural healing capabilities.

Concerns regarding clinical applications of adipose tissue derived stemcells in the medical community are based on the possibility of pathogenand xenogenic pathogen contamination due in part to fetal bovine serumor animal sera used in culture media of cells. Because of the researchgrade techniques used during the handling and processing of tissuesamples, use of the final products would be unsuitable for humanapplication (Preadipocytes in the human subcutaneous adipose tissuedisplay distinct features from the adult mesenchymal and hematopoieticstem cells. Journal of Cellular Physiology 205(1):114-22, 2005 Octoberand Characterization of freshly isolated and cultured cells derived fromthe fatty and fluid portions of liposuction aspirates. Journal ofCellular Physiology 208(1): 64-76, 2006).

There is an immediate and long-felt need for methods and systems for theutilization of human tissue to be suitable for clinical application. Thepresent invention satisfies these needs with respect to adipose tissueand adipose tissue-derived stem cells.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment, the invention is directed to a method ofisolation of stromal vascular derived stem cells which is initiated bysupplying an shipment package including a defined client samplecontainer and thereafter, inspecting shipment package components for (i)integrity of a client sample container containing an adipose tissuesample and (ii) completed recording information, contained therein. Theshipment package components are introduced to a processing module of adatabase via a log-in port by scanning a barcode on the client samplecontainer in the completed recording information. The method continuesby removing collection medium in the client sample container and washingthe adipose tissue sample. Preferably, oil dispersed from the adiposetissue sample is substantially removed and a digestion solution isprepared. The digestion solution is injected into the adipose tissuesample to form a digestion mixture within the client sample container,and incubating the digestion mixture. The incubated digestion mixture isincubated and thereafter centrifuged. Withdrawing a stromal vascularfraction phase of the centrifuged digestion mixture and centrifuging asuspension of the filtered digestion mixture isolates a first stromalvascular pellet. Thereafter, the supernatant of the centrifugedsuspension is removed and the first stromal vascular pellet isre-suspend by trituration in red blood cell lysis buffer forming a cellsuspension. The cell suspension is centrifuged to form a second pellet.The cell suspension of the centrifuged solution is removed, andthereafter, the second pellet is re-suspending by trituration addingsalt solution forming a second suspension. The second cell suspension iscentrifuged to form a third pellet; retaining the supernatant from thethird pellet for a secondary sterility test sample.

In another embodiment the invention is directed to a system forisolation of stromal vascular derived stem cells including a shipmentpackage, a database, at least one sterility test system, a digestionsolution, at least one separation container, viability testing, and astorage facility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of the method of the present invention;

FIG. 2 is a preferred embodiment of the container of the presentinvention;

FIG. 2A is a side view of a complimentary centrifuge insert illustratingthe container in phantom;

FIG. 2B is a plan view of the complimentary centrifuge insert for thecontainer;

FIG. 3 illustrates the system;

FIG. 4; is a plan view of the shipment package of the system; and

FIG. 5 illustrates the sample container of the shipment package and thedigestion solution, sterility test program, at least one separationcontainer and storage container of the system.

DETAILED DESCRIPTION OF THE INVENTION

Certain terminology is used herein for convenience only and is not to betaken as a limitation on the present invention. The terminology includesthe words specifically mentioned, derivatives thereof and words ofsimilar import. The embodiments discussed herein are not intended to beexhaustive or to limit the invention to the precise form disclosed.These embodiments are chosen and described to best explain the principleof the invention and its application and practical use and to enableothers skilled in the art to best utilize the invention.

In a first embodiment, the invention is directed to a method ofprocessing aspirated adipose tissue for the isolation of stromalvascular fraction (SVF) derived stem cells 10. The method is initiatedby supplying a shipment package including a defined client samplecontainer 12. The shipment package can include various components inaddition to the defined container with client information for extractionof the adipose tissue sample by a physician. Thereafter inspecting theshipment package components for (i) integrity of a client samplecontainer containing an adipose tissue sample and (ii) completedrecording information, contained therein 14 is performed at a designatedlaboratory site or other designated facility site.

The design of the client sample container is an important feature forvarious embodiments of the invention of the present application toobtain the desired resultant product(s) in a financially effective andlabor efficient manner. Therefore, the following defines key importantfeatures of the container design and the importance of each feature. Oneskilled in the art would recognize the following description is notintended to be limiting but to encompass the required features.

The client sample containers should be made of material such as ethylvinyl acetate and/or polyethylene, or fluoro-ethylene propylene, orcombination thereof, which does not release or leach any potentiallytoxic substance into stored cells. Fluoro-ethylene propylene (FEP) hasthe potential advantage of being gas permeable, so oxygen can diffuse inand carbon dioxide out, which may improve cell viability during storageand transport. Further, the containers must be FDA-approved for use instorage of blood, cells or blood-derived products to allow for clinicalapplications.

The client sample containers should be sterile and approximately 100 mlto 200 ml in volume so as to contain an adequate adipose tissue (AT)sample and requisite medium which can maintain the viability of theadipose tissue for processing and use (as discussed herein). Thecontainers must have at least three (3) ports; one for adding thecollection medium, one for adding the aspirated adipose tissue, and onefor removing the medium, adding the washes and digestion enzyme, andremoving the SVF fraction. A preferred configuration of the clientsample container would have five (5) ports, three on top, one in thecenter of a tapered bottom, and one to a side of the bottom. At leastone filter is included within the container for separation.

All ports except one should be “female” ports, one port can be for aspike, (e.g. a large-bore, plastic “needle” meant to be inserted onceinto a port and subsequently kept in place located on a lower end of thecontainer so as to allow gravity for draining contents as appreciated bythose skilled in the art. For example, there is a spike on a 40 micronmesh filter used to recover the SVF from the container after digestion.The spike is nearly the diameter of the port tubing and fits in tightly.Since this is the last time the container is used, the integrity of thecontainer after this step is immaterial. All of the ports must have capsto ensure the elimination of contamination.

The tubing on the ports should be no more than 6 inches in length. Longtubing would be a waste of space and material and could cause loss ofsample; e.g., fat could get clogged or adhere in the tube. The portsinclude tubing connecting the port opening with the inside of thecontainer.

Referring to FIG. 2, a preferred example of the container 100 isillustrated with a complementary centrifuge insert 102 (shown inphantom). In the preferred example, the container 100 includes aPhysician port 104A, a medium entrance port 104B, and an oil removingport 104C on a top side 106 and a 3-way port 108 on a bottom side 110.The Physician port 104A, a medium entrance port 104B, and an oilremoving port 104C are color coded to avoid contamination. Moreover, thePhysician port 104A may include a pierced septum for direct connectionto a syringe. In a preferred embodiment, the 3-way port 108 includes aspike port 112 positioned perpendicular to the bottom side 110, asufficient distance from the 3-way port 108 and final filter 110 topreclude interfering with the function of either component.

The container 100 includes a filter 114 extending the width of thecontainer 100, wherein the filter 114 initiates a tapered section 116 ofthe container 100. The tapered section terminates at the 3-way port 108.A second filter 118 traverses the bottom side 110 distal to a terminusend 120 of the spike port 112. The filter is approximately 100micrometer nylon mesh and the second filter is approximately a 40micrometer nylon mesh. The 100 micrometer mesh filter will allowindividual cells of the SVF to pass through, but will exclude large,mature adipocytes and clumps of smaller cells still held together byextracellular matrix material, and will also reduce the potential forclogging the smaller mesh filter below. The 40 micrometer mesh filterwill exclude most immature adipocytes and smaller clumps of cells, butwill allow isolated SVF cells such as adipose stromal cells andendothelial progenitor cells to pass.

Referring to FIGS. 2, 2A and 2B, the complementary centrifuge insert 102has an exterior surface 102A which fits into a standard bucket typecentrifuge such as Beckman GS-6R with a GH 3.8 swinging bucket rotor. Acentral insert section 102B for placement of the container 100 definesan interior portion 102C. The interior surface 102C includes pocketinserts 122 which are positioned to accept the 3-way port 108 at thebottom side 110 of the container 100 (shown in phantom) in a foldedposition as best illustrated in FIGS. 2A and 2B. In the folded position,the dimensions of the container 100 allow the “fold” to act a lockingmechanism to eliminate leakage during centrifuging the sample (asdiscussed herein). The container could have thicker areas or ridgesimmediately above and below the site of the fold to enhance the sealwhen the container is folded, in a fashion similar to a zipper-topcontainer. Alternatively, the container could be tightly sealed afterfolding by attaching a clip similar to a binder clip by sliding orclamping. This could also lock the container into position more securelyinto the centrifuge bucket insert. Thus, centrifuging forces the samplethrough the filter 114 but the position “fold” eliminates leakage.Referring again to FIG. 2A, the insert 102 includes weight adjustmentpockets equally spaced on either side of the central insert section102B.

Again referring to FIG. 1 illustrating the method, after filling withcollection medium, the client sample containers are sealed in outercontainers before shipping. The outer container is to protect the innercontainer, particularly ports which have been accessed, fromcontamination. The outer containers also serve to prevent leakage andcontamination of the shipping container incase the integrity of theinner container is compromised. Outer containers do not have to besterile, and don't have to be of the same material as the innercontainers, because the cells will not come in contact with the outercontainers. The outer containers must be sealable, and can becommercially obtained separately.

Inspecting the shipment package 14 includes ensuring that (a) thecollection container is not past a defined expiration date, (b) theclient sample was collected within the past 48 hours, and (c) therecording information is accurate. If the conditions set in any of a, b,or c are not met, then the sample is not acceptable and must bediscarded as biohazard waste. This discard will be recorded fororganization and “tracking” of the sample.

Most commonly, the shipment package includes a bar-coded, medium-filledclient sample container in an outer container (as discussed herein), asterile, 60 cc syringe, a patient-specific bar-coded shipping containerapproved for biohazardous materials containing an absorbent sheet, aTyvek® outer container, sufficient bubble-wrap to stabilize thecontents, and foam insulation in an outer corrugated cardboard box (thelatter items commercially available from Saf-T-Pak®). The appropriateneedles/cannula and other medical supplies are generally accessibleequipment which will be supplied by the physician, but may be includedas part of the shipment.

The method continues by introducing the shipment package components to aprocessing module of a database via a log-in port by scanning a barcodeon the client sample container in the completed recording information16. The database will be custom-designed to have the ability to complywith the requirements of the American Association of Blood Banks (AABB)standard 6.3 and 21 CFR § 820.30 (FDA Guidance, Jan. 11, 2002, “GeneralPrinciples of Software Validation) using, for example, a commerciallyavailable program such as Microsoft's Access program. The database willinclude but is not limited to, the information obtained from theshipment package to coordinate the client sample with the client; suchas the information included in the patient-specific bar-coded shippingcontainer. This information will also be included in a standardizedform. The database may be organized in modules similar to theorganization in the standardized form, will be searchable, and will beprogrammed to produce all the various forms associated with thisprocess.

At the establishment of an acceptable client sample, one vial each ofcollagenase, neutral protease, and DNase I is removed from a freezer andthawed in a biosafety cabinet for use in a digestion solution.Alternatively, a pre-defined mixture of collagense and neutral proteasemay be used, e.g. Roche Liberase®, which contains both collagenase I andcollagenase II, plus thermolysin, a neutral protease. Thawing at roomtemperature and without assistance supports in the protection of theintegrity and viability of these solutions.

The sample(s) meeting the requirements set forth above, are removed fromthe outer container and gently agitated in the client sample containermanually to re-suspend the fat and any sediment in the medium, andfurther, to ensure that the sterility test samples will berepresentative of the contents. The sample container is wiped, mostcommonly with alcohol to ensure it is not contaminated. Most commonlyspray bottles are used with filter-sterilized 70% ethanol orisopropanol.

The client sample container is “hung” and allowed to stand undisturbedfor approximately five (5) minutes to note the presence of visible bloodand estimate the amount of oil from lysed fat as a fraction of the totaladipose tissue present in the client sample. This observation isrecorded in a defined manner usually on a pre-designed form.Observations such as the amount of oil present will be entered into astandardized form, and thus become part of the database. The completedforms can be printed for backup records. For coordination and ease, theforms will all be fill-able online. It is appreciated that upon log-ininto the system, they will be able to choose the form(s) needed forwhichever process they are performing in collection, storage anddistribution of the client sample, in this case, the digestionprocessing. The bar codes on the containers will be scanned, and theinformation in the barcode will be imported into the form from thedatabase.

Sterility of the AT sample within the client sample container is testedto ensure the quality. The test most commonly includes disinfecting thebottom port prior to removal of a defined amount the collection medium18. The removal via a bottom port allows extraction by gravitationalforce thus eliminating any need for a “pump” etc. Sterility andmicrobial testing is performed by standard commercial systems such asBacT/Alert or similar testing. Specific testing procedures are performedin order to comply with and receive required AABB or other professionalorganization certification and adhere to specific current and future FDArules as applicable.

Sterility samples are kept at room temperature until sent to the testinglaboratory. Recognizing there are only a limited number of ports on thecontainer, the same port is used to remove the collection medium fromclient sample container that was used to obtain the sterility sample.The medium is then discarded. The same port is used because the outerpart of the port could be contaminated. Since all the processing will bedone in a biosafety cabinet, the chances of contamination during theprocessing in the laboratory are less than in the physicians' procedureroom.

The AT sample is washed 20 by disinfecting one of the top ports, mostcommonly by wiping with 70% or sterile alcohol with a swab, and adding adefined amount of salt solution. The defined amount is at least equal tothe volume of adipose tissue sample to wash it effectively. The saltsolution, e.g. Hank's Balanced Salt Solution (HBSS), is added by using a60 cc syringe with an 18 gauge needle, or alternatively, a split septumneedle-free port such as the BD Q-Syte™ Luer access port may be used toadd and remove solutions.

One skilled in the art would recognize ports come in several differenttypes; most must be traversed using either a needle or a spike(basically, a fat plastic needle). An 18 gauge needle is used commonlyas an efficient removable tool. It is understood, the lower the gaugenumber, the larger the needle diameter. While a smaller needle (such as27 gauge) makes a smaller hole, and thus damages the septum less, it isharder to inject or remove the large volumes of medium needed in thedigestion process. All ports for needles have Luer® fittings, as do allthe syringes which are used, which are identical regardless of needlesize.

The client sample container is gently agitated and allowed to standundisturbed for a defined time period most commonly about five minutesUsing the same port that was used to obtain the sterility sample, thewash is removed and discarded. The container is allowed to hangundisturbed again until fat is observed floating in a single layer atthe top of the container, oil (if present) on top of the fat.Alternatively, the container may be briefly centrifuged at a lowrelative centrifugal force, for example, 800 revolutions per minute for3 minutes, in order to separate the phases.

Oil dispersed from the adipose tissue sample is substantially removed22. If there is significant (>about 5 mL) oil on the top of the ATsample; as much of the oil as possible should be removed withoutdisturbing the AT sample by maneuvering the container so that the oil ispositioned by one port; using a syringe and needle complementary to theport. If blood or oil is still visible after the first wash, the ATsample can be washed again with the salt solution as described above,allowing the sample to separate before removing wash buffer; the HBSSwash defined herein.

A digestion solution is prepared 23 as discussed herein and is injectedinto the client sample container having the adipose tissue sample toform a digestion mixture within the client sample container. The outsideof the vials of collagenase, neutral protease, and DNase I, orcombination product thereof, for the digestion solution (thawed aspreviously discussed for use herein), are wiped to ensure sterility,most commonly with an alcohol swab.

Using sterile, 2 ml pipets transfer the solutions to a 50 ml centrifugetube containing 48 ml HBSS pre-warmed to 37° Celsius. The tube is cappedand mixed by gentle agitation of the tube. The basis of this dilutionratio is the concentration and enzymatic activity of the enzyme stocksolutions, which are based on (i.) product protocols (ii.) experience,and (iii.) what is known in the art. In regard to “what is known in theart”, most workers routinely make enzyme solutions as a weight/volumepercentage, or milligrams per milliliter, for example, 0.1 percentcollagenase, or 1 milligram per milliliter, is widely used for digestionof adipose tissue (Growth and differentiation of human adipose stromalcells in culture. In Methods in Molecular Medicine: Human Cell CultureProtocols, 1996, 41-51. An advantage of the protocols developed in theinstant application is to base the dilution on the amount of enzymeactivity, rather than simply weight/volume ratios, since the activity ofdifferent enzyme lots can vary drastically. For example, 1 mg of lot “A”might have one unit of activity, whereas 1 mg of lot “B” could have only0.7 unit of activity. Therefore, a 1 mg/mL solution of lot “B” enzymewould only have 70% of the activity of a 1 mg/mL solution of Lot “A”enzyme, resulting in less efficient digestion. The method of the instantapplication eliminates this variable by basing the enzyme dilution onthe specific activity rather than the mass, making the process morereproducible. This is critical for a commercial process.

The digestion solution is injected 24 into the washed AT sample usingone of the top ports of the container. The digestion mixture isincubated 26 at 37 degrees Celsius for 45 minutes while being agitatedon a rocking platform at about 24 rocks per minute. At the end of thedigestion, the adipose tissue is converted from a suspension of tissuefragments up to 4 millimeters in size into a much smoother suspension inwhich most tissue fragments are less than 1 millimeter in diameter, asmost of the adipose tissue is dissociated into isolated matureadipocytes and stromal-vascular fraction cells, although some whitish,connective tissue may remain intact. Thereafter, the solution iscentrifuged at a low speed to separate the mature adipocytes from therest of the digestion mixture 28.

The stromal vascular fraction phase of the centrifuged digestion mixtureis withdrawn 30 through a sterile, 40 micrometer mesh filter. Thecentrifugation of the digestion mixture in the container serves toseparate the SVF from the adipocytes and undigested adipose tissue. Morespecifically, the first low-speed centrifugation, while the digestionmixture is still in the container, separates the oil and fat from thestromal-vascular fraction. However, the geometry and composition of theproposed container does not allow for a concentrated pellet since thestromal-vascular fraction pellet is very small, typically less than 0.1milliliters, and the container narrows gradually from the full widthabout midway down the container. The stromal-vascular fraction thusspreads out over a fairly large area near the bottom. Removing this SVFfrom the container to a centrifuge tube and re-centrifuging allowsformation of a “tight” pellet at the bottom of the tube (as discussedherein), so that greater than 95 percent, and as much as 99 percent, ofthe enzyme solution can be removed. One skilled in the art wouldappreciate the container plastic (discussed previously herein) permitsgreater adhesion than the rigid polypropylene of which most disposablecentrifuge tubes are made, and significant amounts of debris arereleased from the adipose tissue during digestion, so some of the SVFmaterial is able to adhere to the inside of the container near thebottom

The suspension of the filtered digestion mixture is centrifuged in two50 mL tubes upon removal from the container, isolating the first stromalvascular pellet 32. The supernatant of the centrifuged, filteredsuspension isolated is removed 34. The stromal vascular “tight” firstpellet is re-suspended 36 by trituration in a red blood cell lysisbuffer, eliminating red blood cells, as well as removing residualenzymes and debris, forming a cell suspension which is centrifuged toform a second pellet 38. This serves as another wash, and removeshemoglobin released from lysed red blood cells. The supernatant of thecentrifuged cell suspension is removed 40.

The second pellet is re-suspended by titration adding HBSS forming a“second cell suspension” 42. This second cell suspension can be countedand analyzed for viability using; 1) stains specific for live and/ordead cells and a hemacytometer with a microscope, or 2) a commerciallyavailable automated cell analyzer. In a preferred method of counting andanalyzing the of the second cell suspension, a small aliquot (20microliters) of the second cell suspension is mixed with an equal volumeof a mixture of acridine orange and propidium iodide stains and countedusing the Nexcelom Cellometer Vision instrument (Nexcelom Biosciences).The second cell suspension is centrifuged 44 to form a third pelletwhich is stored in a biosafety cabinet for initiation of acryopreservation process 46. Furthermore, the supernatant of the thirdpellet serves as a secondary sterility test sample which may reveal theelimination of contaminants that may have been present in the initialsample, and ensure that no contamination was introduced during theprocessing in the laboratory.

The “third pellet” defines a stem cell pellet product, e.g. a washed SVFpellet, produced by the method, wherein the stem cell pellet includes amixture of cells of pre-adipocytes, adipose-derived mesenchymal stemcells, microvascular endothelial cells, endothelial progenitor cells,monocytes, and small numbers of vascular smooth muscle cells. Themixture must contain no mature adipocytes, and at least 1% of thenucleated cells in the mixture must be adipose-derived mesenchymal stemcells. The mixture or “stem cell pellet product” or “washed SVF pellet”must exhibit a combined viability by acridine orange/propidium iodide ortrypan blue dye-exclusion assay of no less than 35%. Further, theadipose-derived mesenchymal stem cells contained therein must be capableof proliferation when placed in contact with a suitable culture mediumunder appropriate environmental conditions known to those skilled in theart of cell culture.

In another embodiment, the invention is directed to a system forisolation of stromal vascular derived stem cells. Referring to FIG. 3,the system 210 includes a shipment package 212, a processing system 214,at least one storage facility 216 and a database 218. The database 218stores information obtained from the shipment package 212, processingsystem 214, and at least one storage facility 216.

Referring to FIG. 4, the shipment package 212 includes a bar codedclient sample container 220, a sheet of absorbent material that must becapable of absorbing all of the fluid in the sample (not shown), andshould be Department of Transportation/International Air TransportAssociation] approved for shipping biological or medical samples. Asecond container 222, most commonly a zipper-top plastic container tocontain the client sample container and the sheet of absorbent material,and a form of recording information 224 are also included. The thirdcontainer 225 contains the bar coded client sample container 220 for thereturn shipment to the processing laboratory and is most commonly, acommercially available Saf-T-Pak® container including a plasticcontainer and may include a Tyvek® container.

The form of recording information 224 works in tandem with the database218. The database 218 includes an encoded program to organize and storeinformation regarding the sample and recording information and iscustomized to the specific requirements for the coordination of samplesto the client. The database 218 will include all data, not just forstorage, identification and distribution, and further informationobtained from the second bar coded container 222. In addition, thedatabase 218 will include information regarding preparation of allreagents.

Referring to FIG. 5, the processing system 214 includes at least onesterility test system 226, a digestion solution 228, and at least oneseparation container 230. Each component of the processing system 214has been described here in regard to the method embodiment herein. Asbest illustrated in FIG. 5, the processing system 214 components work ina coordinated manner with other elements of the system 210.

The sterility test system 226 includes a combination of oneBacT/Alert-iAST sample bottle and one BacT/Alert-iNST incubated atdesignated temperature(s) in the BioMérieux BacT/Alert system. It isrecognized that other sterility tests could be used including fluidthioglycollate medium, soybean-casein digest medium, and blood agarplates incubated at the appropriate temperature(s). All the test bottlesin the lab will be inoculated and either tested in-house on theBioMérieux BacT/Alert system or sent out for such testing. All sterilitytest cultures must be incubated at 30°-37° C. for fourteen (14) days,except for soybean-casein digest medium which may be incubated at 20-25°C., for a minimum of 14 days and inspected on days 3, 4, or 5, days 7 or8, and day 14 for turbidity (broths) or colony formation (blood agarplates). Results of each observation will be recorded, most commonlypursuant to 21 CFR § 610.12.

The present invention will now be described based on the followingexamples.

Example 1

Step Action 1 Upon receipt, inspect the shipment for (a) the clientsample container(s) and (b) the completed form. Ensure that theintegrity of the sample container(s) is intact. If the sample containeris not bar-coded, or if there is any evidence of leakage from thecontainer(s), the sample is not acceptable and must be forwarded toquality management and the Laboratory supervisor notified. 2 Printlabels matching the barcode on the sample and place on the (2) 50 mLtubes, two (2) 15 mL tubes, and one (1) 12 mL tube required. Print fouradditional labels for the batch record and the three CellSeal vials. Atleast two of the labels must be in a wrap-around format and one longformat. 3 Remove one vial of collagenase MNP-S from the −80° C. freezerand thaw in biosafety cabinet (BSC), and place one vial of DNase I fromthe −20° C. freezer in the BSC. 4 Remove the sample(s) meeting therequirements set forth in 1 and 2 above, from the overwrap and gentlyrock the container manually three times. Weigh the container on thetop-loading scale and record the weight. Wipe the sample container with70% alcohol, hang the container from the IV bar in the biosafetycabinet, and allow to stand undisturbed for ten (10) minutes. Note thepresence of visible blood and estimate the amount of oil from lysed fatas a fraction of the total adipose tissue (AT) present in the sample,and record. 5 Snap the cap off the needle access port, wipe the portwith a 70% alcohol swab and remove 20 mL of the collection medium usinga 20 cc syringe with an 18 gauge needle. Inoculate 10 mL into oneBacT/Alert - iAST sample bottle and 10 mL into one BacT/Alert - iNST forphysician sample sterility testing. These are kept at room temperature(20°-25° C.) until sent to the testing laboratory. 6 Using the Q-Syteport, remove the remaining collection medium using an 18 gauge needle ona 60 cc syringe and discard. Re-weigh the container, record the weight,wipe with 70% alcohol, and return to the BSC. Wipe the Q-Syte port witha 70% alcohol swab and add 50 mL of Hank's Balanced Salt Solution (HBSS)using a 60 cc syringe. Gently rock the container manually three times byhand and centrifuge at 800 rpm for three (3) minutes. 7 Return thecontainer to the BSC and hang on the IV bar. Wipe the Q-Syte port with a70% alcohol swab, attach a 60 cc syringe and remove and discard the washsolution. Re-weigh the container, record the weight, wipe with 70%alcohol, and return to the BSC. 8 Wipe the outside of the vials ofcollagenase and DNase I with a 70% alcohol swab. Using a sterile, 2 mlpipet, transfer the collagenase solution to a 50 ml centrifuge tubecontaining 49 ml HBSS pre-warmed to 37° C., rinse the vial with 2 mL ofthe solution and add the rinse to the tube. Then add 0.2 mL (200 μL) ofthe DNase I stock to the tube using a micropipettor, cap the tube andmix by gently swirling the tube. This is the digestion solution. 9 Propthe container against the tube rack with the ports pointing up above therack. Wipe the Q- Syte needle-free access port with a 70% alcohol swab.Remove the plunger from a sterile 60 cc syringe and attach the syringeto the Q-Syte port. Pour the digestion solution into the syringe, andthen open the clamp on the container port to allow all of the solutionto enter the container. Close the clamp on the container and remove thesyringe. 10 Fold the container over at the bottom where the ports enterthe container and secure with a rubber band. Place the digestioncontainer on the rocker platform in the 37° C. incubator and incubatefor 45 minutes at 24 rocks per minute. 11 During the digestion period,the technician may log-in and perform steps 1-10 of a second AT sampleif necessary to complete processing of all samples on the date received.12 After digestion is completed, remove the container from the incubatorback to the Biosafety cabinet and add another 50 mL of HBSS via theQ-Syte port using a sterile 60 cc syringe. Rock the container manually 3times and place in the blood container cup in the centrifuge. Ensurethat there are no tight creases in the container, that the container isstably in position, and that the central, bottom port is centered in thebottom of the bucket, and then put on the bucket cover. Ensure that therotor is balanced by placing the bucket on the beam balance against theopposite bucket containing a sham sample container, and adjusting themass of the balance bucket by adding or removing water from the shamcontainer or a centrifuge tube in the bucket as needed. Centrifuge at800 rpm for 3 minutes. 13 Remove the digestion container from thecentrifuge bucket, taking care not to disturb the separated phases, andhang in the biosafety cabinet. Remove the cap from the third, unusedmale luer port and attach a sterile 40 μm mesh filter to the port.Gently massage the lower portion of the container to release any cellsdiffusely pelleted on the container. 14 Loosen the caps on the twolabeled 50 mL tubes, remove the cap from the spike at the end of thefilter, and drain the bottom phase containing the stromal-vascularfraction (SVF) through the filter into two (2) 50 mL centrifuge tubes.15 Ensure that the tubes balance, and then place the tubes in opposite50 ml tube buckets. Centrifuge at 1,200 rpm for 10 minutes. 16 Removethe tube(s) from the centrifuge bucket, taking care not to disturb theSVF pellet, and place in the biosafety cabinet. Using an aspiratingpipet attached to the vacuum set-up, remove as much of the supernatantsas possible (all but ≤1 ml). Re-cap the tubes, flick the bottom of thetubes to loosen the SVF pellet, and add 10 ml of ACK lysis buffer to onetube using a 10 ml pipet. 17 Using the same pipet, re-suspend the pelletby gentle trituration, then transfer the suspension to recover thesecond pellet and transfer to the labeled 15 ml centrifuge tube. Placethe tube in the 15 ml tube bucket and balance the rotor. Centrifuge at1,200 rpm for 10 minutes. 18 Aspirate the supernatant, re-cap the tube,flick the bottom of the tube to loosen the SVF pellet, and add 10 ml ofHBSS using a 10 ml pipet. Triturate to produce a homogeneous suspension,remove a 25 μl sample using a sterile tip on a P20 Pipetman and add to a0.65 mL microcentrifuge tube containing 25 μL of AO-PI Live-Dead stain.Set the centrifuge to 10° C., then place the tube in the bucket oppositea balance tube, but do not start the centrifuge. 19 Load a Cellometerslide with 20 μL of the cell suspension/AO-PI mixture. Insert theCellometer slide into the instrument and determine the cellconcentration and percent viability. Save the data files, then calculatethe total viable cell yield and enter all data into designated form. 20If the Cellometer count is valid, go to step 24. If the Cellometer countis not valid, go to step 21. 21 If the Cellometer count is not valid(i.e., if the count is <1.5 × 10⁵ cells/ml), centrifuge at 1,200 rpm for10 minutes. Remove the tube(s) from the centrifuge bucket, taking carenot to disturb the SVF pellet, and place in the biosafety cabinet. Usinga 10 ml pipet, carefully remove the supernatant and transfer to theclean labeled tube. Inoculate about 4 mL of this into one Bact/Alert -iAST sample bottle and about 4 mL into one Bact/Alert - iNST for processsterility testing. Then flick the bottom of the tube to loosen the SVFpellet, add 5 ml of HBSS using a 10 ml pipet and triturate to re-suspendthe cells. Remove a 25 μl sample using a sterile tip on a P200 Pipetmanand place in 25 μL of AO-PI, then load a Cellometer slide and recount.If the count is still less than 1.5 × 10⁵ vc/mL, the yield is inadequatefor cryopreservation of a client sample. 22 If the re-count is valid,place the tube(s) in the 15 ml tube bucket and balance the rotor. Setthe centrifuge to 10° C., then place the tube in the bucket opposite abalance tube and centrifuge at 1,200 rpm for 10 minutes. 23 Remove thetube(s) from the centrifuge bucket, taking care not to disturb the SVFpellet, and place in the biosafety cabinet.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A method of isolation of stromal vascular derived stem cellscomprising the steps of: a. supplying a shipment package comprising adefined client sample container having a first filter and a secondfilter; b. inspecting the shipment package components for (i) integrityof the client sample container containing an adipose tissue sample andcollection medium and (ii) completed recording information, containedtherein; c. introducing the shipment package components to a processingmodule of a database via a log-in port by scanning a barcode on theclient sample container in the completed recording information; d.testing the sterility of the adipose tissue sample from the clientsample container; e. removing the remaining collection medium in theclient sample container; f. washing the adipose tissue sample; g.preparing a digestion solution by diluting an enzyme stock solutionbased on the amount of enzyme activity; h. injecting the digestionsolution into the sample container containing the adipose tissue sampleto form a digestion mixture within the client sample container; i.incubating the digestion mixture; j. centrifuging the incubateddigestion mixture; k. withdrawing a stromal vascular fraction phase ofthe centrifuged digestion mixture, wherein the stromal vascular fractionconsists of the fluid phase below the floating mature adipocytes and oilfrom lysed adipocytes; l. centrifuging a suspension of the digestionmixture in order to isolate a first stromal vascular pellet from thedigestion solution; m. removing supernatant of the centrifugedsuspension isolated in l; n. re-suspending the first stromal vascularpellet by trituration in red blood cell lysis buffer forming a cellsuspension; o. centrifuging the cell suspension to form a second pellet;p. removing supernatant of the centrifuged solution isolated in o; q.re-suspending the second pellet by trituration adding salt solutionforming a second suspension; r. centrifuging the second cell suspensionto form a third pellet—comprising a mixture of cells of pre-adipocytes,adipose-derived mesenchymal stem cells, microvascular endothelial cells,endothelial progenitor cells, and monocytes, wherein the mixture ofcells either i) does not contain mature adipocytes, and demonstrates acombined viability by acridine organge/propidium iodide or trypan bluedye-exclusion assay of no less than 35%, or ii) at least 1% of thenucleated cells in the mixture are adipose-derived mesenchymal stemcells; s. retaining the supernatant from the third pellet for asecondary sterility test sample; and t. proliferating theadipose-derived mesenchymal stem cells of the third pellet in a suitableculture medium under appropriate environmental conditions.
 2. The methodof claim 1, wherein the sample container comprises three female portsdisposed on a top portion of the sample container and one three-way portdisposed at a bottom of a tapered section of the sample container, thethree-way port including a spike port positioned perpendicular to abottom side of the sample container, and where each of the portscomprises a removable cap, wherein the first filter is position betweenthe top portion and the tapered bottom of the sample container and thesecond filter is in the center of the tapered bottom proximal to theport located in the center of the tapered bottom, wherein the methodfurther comprises filtering the digestion mixture via the first filterto allow individual cells of the stromal vascular fraction phase to passthrough but excluding large mature adipocytes and smaller cells heldtogether by cellular matrix, wherein the filtering of the digestionmixture initiates at step h. and terminates at step j.
 3. The method ofclaim 2, wherein inspecting the shipment package comprises: ensuringthat (a) the sample container is not past a defined expiration date, (b)the adipose tissue sample was collected within the past 36 hours, and(c) the recording information is accurate.
 4. The method of claim 3,wherein the method further comprises testing the sterility of theadipose tissue sample after step b.
 5. The method of claim 4, whereinpreparing a digestion solution comprises the steps of: combiningcollagenase, neutral protease, and DNase I with a salt solutionpre-warmed to 37° C.
 6. The method of claim 5, wherein the methodfurther comprises removing a sample of the re-suspended sample of stepm. and calculating the total viable cell yield and recording results ofthe total viable cell yield.
 7. The method of claim 6, wherein themethod further comprises testing sterility of the supernatant obtainedin step p.
 8. The method of claim 7, wherein the sample container has atleast one port on an upper portion of the sample container and at leastone port on a lower portion of the sample container.
 9. The method ofclaim 8, wherein the sample container is made of a material which doesnot release or leach any potentially toxic substance into stored cells.10. The method of claim 9, wherein the sample container is made of amaterial selected from the group consisting of ethyl vinyl acetate,polyethylene, fluoro ethylene propylene and combinations thereof. 11.The method of claim 10, wherein the sample container is sterile andapproximately 100 ml to 200 ml in volume.
 12. The adipose-derivedmesenchymal stem cells obtained by the method of claim 1 or
 2. 13. Theadipose-derived mesenchymal stem cells of claim 12 which comprisephenotypes CD14−, CD19−, CD29+, CD31−, CD34−, CD44+, CD45−, CD49d+,CD73+, CD90+, CD105+, and CD146+, the phenotypes exhibited on 100% ofthe cells.
 14. The adipose-derived mesenchymal stem cells of claim 13,which consistently differentiate into Oil Red+ adipocytes, Alcian Blue+chondrocytes, and Alizarin Red+ osteocytes.